GLUCOCORTICOID RECEPTOR IN THE EARLY LIFE PROGRAMMING OF CARDIAC RESILIANCE

In the time shortly before and after birth (the first few months of life), the vital organs, including the heart and the lungs, undergo remarkable changes that are essential for the baby to survive once it is born. The changes that occur in the heart before birth set the foundations for later life, and influence the risk of developing heart disease in adulthood. Glucocorticoids, an important class of steroid hormone produced naturally by the body, play a vital role in these maturational changes. We utilise this clinically in premature babies (or when premature birth appears likely) with the routine administration of potent man-made glucocorticoids to improve lung function and neonatal survival. This treatment, termed antenatal corticosteroid therapy (or ACT), has been assumed to be very safe for pregnant women and their unborn babies. However, there is convincing evidence from animal studies and in humans, that prolonged exposure to higher than normal levels of glucocorticoids before birth predisposes an individual to a host of later life diseases, including heart disease. The reasons why are incompletely understood, but may reflect a wrongly timed or excessive response to the normal maturational effects of glucocorticoids.
To be able to understand the harmful effects that glucocorticoids are capable of and which have their origins in the time before birth, we need to know what they do normally before birth in the heart. Glucocorticoids switch genes on or off by interacting with specific glucocorticoid receptors that allow them to initiate responses in target tissues. We created mice that are genetically altered to lack glucocorticoid receptors in just the muscle cells of heart and blood vessels to find out what glucocorticoids do in these cells. We found that if glucocorticoids cannot act in these muscle cells, the heart appears grossly normal but the muscle fibres in heart cells appear immature and the heart does not contract as it should. This might be why up to half of these mice die shortly after birth. We found that glucocorticoids help heart muscle cells to generate energy using oxygen and that this is important for glucocorticoids to improve the structure of muscle fibres. This is intriguing, as the same oxygen-utilising systems stop heart muscle cells multiplying shortly after birth. This suggests that glucocorticoids help prepare, or mature the heart, ready for birth, but at the same time regulate the number of cells in the heart after birth. Fewer muscle cells because of too much glucocorticoid before birth is likely to make the adult heart vulnerable in situations where it has to work harder.
We now plan to test our idea. First, we will test if the same oxygen-ultising systems increased by glucocorticoids help mature other properties of heart muscle cells, such as contraction. Next, we will ask whether ACT in mice alters the timing and extent to which heart muscle cells multiply before and shortly after birth. We expect to see the opposite effect on heart muscle cell multiplication in our mice that lack glucocorticoid receptors in these cells. Finally, we shall ask if we can 'rescue' the hearts of our genetically altered mice by putting back glucocorticoid receptors just in heart muscle cells. Importantly, we shall do this in a way that allows us to switch the receptors off as soon as the mice are born, or later during the period immediately after birth. This will allow us to test if the different effects of glucocorticoids upon the heart, manifest in newborns and adults and including any effects on cell number, all have their origins in prenatal life.
This research will provide important new understanding of the benefits and risks of antenatal glucocorticoid actions upon the immature heart. It will enable us to establish, in theory, areas for future refinement in therapy, for example, to avoid over treatment of babies and to limit adverse effects, whilst optimising the benefits of treatment.

Glucocorticoids are essential to mature fetal organs and tissues in preparation for life after birth, the reason they are used clinically in pregnant women at risk of preterm delivery. Conversely, excessive prenatal exposure to glucocorticoid increases risk of cardio-metabolic disease in adulthood. We have used glucocorticoid receptor (GR) knockout mouse models to show a previously unappreciated and essential role for GR to functionally, structurally and biochemically mature the fetal heart. Experiments in primary fetal mouse cardiomyocytes suggest that this occurs through remodelling of mitochondria. Mitochondrial mechanisms underlie the perinatal switch in cardiomyocytes from hyperplastic growth to cell cycle exit, binucleation and hypertrophic growth. Pilot data in our 'SMGRKO' mice that lack GR selectively in cardiomyocytes and vascular smooth muscle point to a delay in this switch, resulting in greater cardiomyocyte endowment in adulthood. This suggests that glucocorticoid induced maturation of the fetal heart is coupled to, and at the expense of, cardiomyocyte endowment in adulthood. To test our hypothesis, we will (i) establish if mitochondrial mechanisms underlie all aspects of glucocorticoid-induced maturation of cardiomyocytes, supporting a coupled mechanism behind glucocorticoid effects on cardiomyocyte maturation and growth; (ii) investigate whether prenatal administration of dexamethasone (used clinically) reduces cardiomyocyte proliferation and promotes cell cycle exit and binucleation in vivo and (iii) restrict GR expression in cardiomyocytes to the fetal period to test whether this promotes both cardiomyocyte maturation and exit from the cell cycle, confirming GR dependent mechanisms. If similar mechanisms operate in human fetal development, then appropriate understanding will highlight areas for improvements in therapy, for example to avoid overtreatment of babies and to limit adverse effects, whilst optimising the benefits of treatment.

The most important impact of this research will be in its relevance to pregnant women and their children, particularly those at risk of preterm birth. These women are routinely given antenatal corticosteroid therapy (ACT, potent synthetic glucocorticoids) to improve neonatal survival in the event of premature birth. However, whilst potentially life-saving in premature babies, there is increasing recognition of, and concern over, the risks associated with ACT and its long-term effects on health. Around half of pregnant women that receive ACT in anticipation they might deliver preterm, actually go on to deliver their babies at term. This suggests widespread overtreatment of unborn babies with ACT. Moreover, examination across a wide range of outcomes, including later susceptibility to heart disease, has prompted some to question whether the treatment is as safe as previously assumed. Our research will provide important new information on both the cardiac benefits and risks of ACT. It will therefore impact upon the current debate surrounding the use and benefits of ACT.
Impact will be two-fold in this area. Firstly, in premature babies. Our research will provide insight into the processes by which glucocorticoids normally mature the fetal heart, an area that has not received much attention to date, but which may be very important in the benefits of ACT upon neonatal survival of premature babies. It could identify new biomarkers of cardiac maturity in humans, before and after birth, that can be assessed along with other outcomes such as lung maturity. Indeed, we are currently planning a study in humans to test effects of ACT upon isovolumetric contraction time, a biomarker of glucocorticoid action identified in our preclinical research. A deeper understanding of how glucocorticoids mature the fetal and neonatal heart is also needed to inform optimisation of therapy for maximum benefit, (timing, dosing regime, type of corticosteroid).
Secondly, this research will be relevant to term babies who received ACT. Excessive glucocorticoid exposure in utero increases risk of cardiac disease in adulthood, though the mechanisms remain unclear. Our research will provide mechanistic insight into the developmental origins underlying the increase in susceptibility to heart disease that is associated with ACT. If we discover that the maturational effects of glucocorticoids in the fetal heart are coupled to a decrease in cardiomyocyte endowment in the adult heart, this will have significant relevance for ACT. It will impact future trials examining timing and dosing regime for ACT. It will also provide greater impetus to improve the criteria for intervention in pregnancy to reduce overuse.
Aside from ACT, evidence suggests that the programming effects of stress and poor diet upon future risk of cardiovascular disease involve excessive fetal glucocorticoid exposure. Pregnant women are anxious to ensure the future health of their unborn child. By increasing our knowledge of the developmental windows of susceptibility to the programming effects of glucocorticoids upon heart, women can take steps to appropriately manage diet and stress during pregnancy.
This research will also impact preclinical research in the areas of fetal medicine and regenerative medicine. Our 2 recent publications are already attracting citations (24, to date), indicating impact in these fields. The proposed research will continue to inform preclinical research in sheep (collaborator, Sarah Stock), which now includes examination of glucocorticoid effects on fetal heart. Our research will provide new information about glucocorticoid effects on proliferative (and hence, regenerative) capacity of the neonatal heart and could impact future research in this area. Mice created through this research will be a valuable resource for future research in this area, generating impact.